https://orcid.org/0000-0002-7239-4577
Correction
5 Mar 2026: The PLOS One Staff (2026) Correction: Streptococcus pneumoniae upregulates Toll2, Toll9, and defensin genes in Bombyx larvae infection model. PLOS ONE 21(3): e0344379. https://doi.org/10.1371/journal.pone.0344379 View correction
Figures
Abstract
Pneumococcal diseases caused by the human pathogenic bacterium Streptococcus pneumoniae are a major public health concern worldwide. In this study, we examined the pathogenicity of a clinical isolate of S. pneumoniae in the silk moth, Bombyx mori, larvae infection model. The whole genome sequencing of a clinical isolate of S. pneumoniae, Spn1 identified the presence of genes responsible for its virulence and antibiotic resistance. Spn1 infection of Bombyx larvae resulted in death within 24 h concomitant with an increase of phenoloxidase activity in the hemolymph. The bacterial load increased in the hemolymph within 9 h post-infection (p.i.) Ampicillin, ceftriaxone, tetracycline, imipenem, and erythromycin showed therapeutic effect in infected larvae, although the bacterial strain was resistant to erythromycin in vitro. The Bombyx homologs of mammalian TLR2 and TLR4, known as BmToll2 and BmToll9 (BmToll9−1 and BmToll9−2 isoforms), were upregulated in both the fat body and trachea. The antimicrobial peptide (AMP) genes, BmdefensinA and BmdefensinB, known to be regulated by the Toll signaling pathway, were significantly upregulated in both fat body and trachea after S. pneumoniae infection through hemolymph. Our data indicate that the Bombyx larvae can be a suitable infection model to study the pathogenicity of S. pneumoniae.
Citation: Chowdhury FR, Hossain MI, Jepu TA, Saleh NUA, Zohora FT, Saleh TA, et al. (2026) Streptococcus pneumoniae upregulates Toll2, Toll9, and defensin genes in Bombyx larvae infection model. PLoS One 21(1): e0341929. https://doi.org/10.1371/journal.pone.0341929
Editor: Chikara Kaito, Okayama University, JAPAN
Received: November 4, 2025; Accepted: January 13, 2026; Published: January 30, 2026
Copyright: © 2026 Chowdhury et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The whole genome shotgun project of Streptococcus pneumoniae Spn1 has been deposited at GenBank under the accession number JBBMWB000000000 (https://www.ncbi.nlm.nih.gov/nuccore/JBBMWB000000000.1/).The associated BioProject and BioSample accession numbers are PRJNA1093433 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1093433) and SAMN40653825 (https://www.ncbi.nlm.nih.gov/biosample/SAMN40653825), respectively. Reference genome: Streptococcus pneumoniae strain Hu17, GenBank accession: CP020549.1 (https://www.ncbi.nlm.nih.gov/nuccore/CP020549.1).
Funding: The funder provided support in the form of salaries for authors [MSH], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Streptococcus pneumoniae, known as the pneumococcus, is a Gram-positive, facultative anaerobic, pathogenic bacterium primarily responsible for causing community-acquired pneumonia and meningitis in children under five years old, as well as in elderly people and those with pre-existing health issues. It is estimated that about one million children die worldwide of pneumococcal disease every year, most of whom are young children in developing countries [1].
S. pneumoniae is commonly found in normal flora of the upper respiratory tract, typically colonizing the respiratory tract, sinuses, and nasal cavity [2]. It is a significant bacterial agent responsible for various diseases, including otitis media, sepsis, and sinusitis, as well as community-acquired pneumonia and meningitis [3]. This condition is the predominant cause of complicated pneumonia in pediatric populations [4,5]. The use of antibiotics or vaccines is the two main methods to prevent S. pneumoniae infection. Although vaccination against S. pneumoniae can be an effective way to prevent pneumococcal infection, antibiotics are essential in reducing bacterial load [6]. S. pneumoniae has a remarkable capacity to acquire exogenous DNA from other pneumococci or related streptococci to become antibiotic-resistant and evade vaccine-induced immunity [7]. The unregulated prescription of antibiotics, self-administration of antibiotics, inadequate hygiene in countries with low- and middle-income, and excessive use of antibiotics in agriculture are all factors contributing to the rise of antibiotic-resistant strains [8,9].
Before being used in human trials, novel antibiotics should be tested for their effectiveness and possible adverse effects in a suitable animal model. The utilization of prevalent mammalian model species, like mice, in comprehensive studies aimed at screening for novel antibiotics that can combat newly emerging antibiotic-resistant bacterial strains presents significant financial challenges. There are also ethical considerations linked to the use of mammalian organisms. The Bombyx larvae have been used as an infection model for microorganisms pathogenic to humans [10,11]. The Bombyx larvae infection model is a useful to study not only host-pathogen interaction but also the therapeutic effect of antibiotics on a range of bacterial classes, including Staphylococcus aureus, Escherichia coli O157:H7, Klebsiella pneumoniae, Niallia circulans, and Klebsiella aerogenes [12–16]. This infection model has also been used to study fungal pathogenicity [17]. The prospect for discovering novel drugs effective against antimicrobial resistant (AMR) bacteria is also high according to the recent reports [18]. The fact that Bombyx larvae and mammals have comparable pharmacokinetics of antimicrobial agents makes it an attractive infection model to screen for antibiotics against bacterial pathogens [19].
Although insects apparently lack adaptive immunity, remarkable similarities have been observed with respect to immune response between insects and mammals [20,21]. Insect hemocytes or immune cells of blood recognize pathogens, phagocytose them, and kill the ingested microbes with superoxide production that is similar to neutrophils [22]. Even though there are some clear differences, there are a lot of similarities in how insects and humans encode the antimicrobial peptide (AMP) gene and how harmful bacteria turn on the Toll and Imd signaling pathways [23]. The Toll signaling pathway was first identified in the fruit fly, Drosophila melanogaster, which is essential for the dorso-ventral axis formation in the embryo [24], and later studies revealed its role in innate immunity, including activation of expression of AMP genes [25]. Around ten Toll-like receptors (TLRs) have been identified in mammals, and these transmembrane proteins act as pattern recognition receptors (PRRs) to detect the pathogen-associated molecular patterns (PAMPs) found on the surface of microbial pathogens [26–28]. These TLRs are important to fight off the invading microbes and prevent extensive tissue damage [29]. Among these TLRs, TLR2 and TLR4 primarily mediate the inflammatory responses upon infection by S. pneumoniae [30–32]. The lepidopteran insect, the silk moth, Bombyx mori, has 14 Toll genes, and among those, BmToll2 and BmToll9 are homologs of the mammalian TLR2 and TLR4 [33–35]. Two Bombyx BmToll9 genes, BmToll9−1 and BmToll9−2, have been identified recently [36,37].
Pathogenicity of S. pneumoniae has been reported in D. melanogaster, the tobacco horn moth, Manduca sexta, and the wax moth Galleria mellonella [38–40], however this study did not address the impact of the bacterium on the expression of homologs of mammalian TLR2 and TLR4 in these species. This research evaluated the efficacy of Bombyx larvae as an infection model for S. pneumoniae, aimed at both antibiotic screening and the examination of the evolutionary conservation of the Toll signaling system in the immune response to this significant human pathogen.
Materials and methods
Growth conditions of bacterial strains
In this research study, four clinical isolates of S. pneumoniae (labeled as Spn1, Spn2, Spn3, and Spn4) were used. The Spn1 strain was a kind gift from Dr. Samir K. Saha of Dhaka Shishu Hospital, and the other three strains were obtained from diagnostic centers of Dhaka, Bangladesh. Standard biochemical tests were conducted for Spn1 (S1 Table) and the other three strains. The AMR profile was established for Spn1 (S2 Table). Initially, the identity of the S. pneumoniae strains has been verified using 16S rRNA gene sequencing. S. pneumoniae strains were routinely grown on Brain Heart Infusion (BHI) agar supplemented with 5% defibrinated sheep blood at 37°C under aerobic conditions. For liquid culture, S. pneumoniae was cultivated aerobically at 37°C in PPB medium, composed of 20 g tryptone, 10 g yeast extract, 2 g glucose, 5 g NaCl, and 2.5 g Na2HPO4 per liter, with a pH of 7.5 as described previously [39].
Whole-Genome Sequencing of Spn1 and bioinformatics analyses
To isolate genomic DNA from S. pneumoniae strain Spn1, the QIAamp® DNA Mini Kit (Qiagen, Hilden, Germany) was utilized according to the manufacturer’s guidelines. DNA quantity and quality were verified with Quantus Fluorometer (Promega) and NanoDrop 2000c (Thermo Fisher Scientific). Sequencing libraries were prepared with Illumina DNA prep kits, and paired-end sequencing was performed on an Illumina NextSeq 550 platform with a High Output Kit v2.5 (300 cycles). The raw sequencing reads were processed by trimming adapter sequences, and data quality was assessed using FastQC (v0.74) on the Galaxy Europe platform. The draft genome was assembled with SPAdes (v3.15.4), and functional annotation was performed using PROKKA (v1.14.6). The NCBI accession number of the draft genome of S. pneumoniae strain Spn1 is JBBMWB000000000. MLST typing was performed using the Streptococcus pneumoniae scheme implemented in the MLST tool (https://github.com/tseemann/mlst). The same typing scheme available at the Center for Genomic Epidemiology (CGE) was also used to confirm allele assignments. The genome was annotated with the RAST toolkit (RASTtk). Plasmid replicons and virulence genes were identified using PlasmidFinder 2.1 [41] and the Virulence Factor Database (VFDB) [42], respectively. Screening for antimicrobial resistance (AMR) genes was conducted using the Comprehensive Antibiotic Resistance Database (CARD) [43], AMRfinder [44], NCBI, and Resfinder [45]. PHASTER was used to identify prophages. A phylogenetic tree was constructed through TYGS using S. pneumoniae strain Spn1, employing the Generalized Time Reversible substitution model, with iTOL utilized for visualizing the tree [46]. To search for any CRISPR-Cas systems in the Spn1 genome, we used the CRISPRCasFinder tool [47], and the analysis was performed with default parameters on the web server.
B. mori strain and rearing conditions
The Nistari-M strain of B. mori was reared at the Bangladesh Sericulture Research and Training Institute (BSRTI). Fresh mulberry leaves were fed thrice a day to the larvae, and they were kept at a temperature of 25 ± 1°C with 80% relative humidity and 16 h light: 8 h dark cycle. Experiments were conducted using larvae on the second or third day of the fifth instar.
Bacterial injection, proliferation, hemocyte viability test, and phenoloxidase activity assay in B. mori larvae
For infecting larvae, an overnight culture of S. pneumoniae was used as described by our group previously [16]. Briefly, larvae (n = 10) were grouped together in boxes that were stored at 25 ± 1°C, 80% relative humidity and 16 h light: 8 h dark cycle. Feeding was stopped 2 hours before injection and the larvae are kept in fasting condition throughout the infection period. Larvae received injections of a specified quantity of bacteria suspended in PBS, delivered into an abdominal leg by means of a 30-gauge (30G × 5/16”). The number of proliferated bacteria in the hemolymph was determined through serial dilution and spread plating, and hemocyte viability was assessed using the Trypan Blue cell viability assay with a hemocytometer. Additionally, the hemolymph phenoloxidase (PO) activity assay was performed as previously described [15].
MIC of antibiotics and ED50 determination in Bombyx larvae
To determine if B. mori larvae are a suitable infection model for testing antibiotic efficacy against S. pneumoniae, the following drugs were used: tetracycline (Sigma-Aldrich, USA), ceftriaxone (Radiant Pharmaceuticals, Bangladesh), ampicillin (Opsonin Dhaka, Bangladesh), imipenem-cilastatin (Incepta Pharmaceuticals Ltd., Bangladesh), and erythromycin (Square Pharmaceuticals Ltd., Bangladesh). Each antibiotic was diluted with distilled deionized water to four concentrations: 50, 5, 0.5, and 0.05 µg g ⁻ ¹ body weight. Larvae received a single 50 µL dose of antibiotic 10 minutes post-infection. No toxicity was observed for the drug-alone treatment at the highest doses of antibiotics used in the uninfected larvae. The minimum inhibitory concentration (MIC) of each antibiotic against S. pneumoniae was ascertained utilizing a Promega GloMax Explorer microplate reader, following Clinical and Laboratory Standards Institute (CLSI) criteria, as previously delineated [16]. The median effective dosage (ED₅₀) was determined using accepted methodologies [12].
Topical application of S. pneumoniae on Bombyx larvae
To infect the silkworm trachea (airway system) through spiracles, we topically applied the indicated number of Spn1 bacteria suspended in 50 µL of PBS onto 10 abdominal spiracles (5 µL drop per spiracle across 5 spiracles on each side) using micropipette, whereas the control larvae were treated with PBS. Larvae were manually restrained without anesthesia during application and were kept isolated for 10 minutes before transferring to the boxes to prevent cross-contamination or oral exposure. RT-qPCR analysis was performed by isolation of RNA from tracheal bushes collected from dissected larvae after 6- and 12 h of infection. Bacterial numbers in hemolymph and tracheal bushes after topical application were determined as described previously [15].
RT-qPCR analysis of BmToll2, BmToll9−1, BmToll9−2, and AMP genes
Gene expression analysis of adipose tissue and tracheal tissues was carried out through RT-qPCR, as previously published [15]. The total RNA was extracted from the fat body in the dorsolateral region of larvae or tracheal bushes from all over the body using TRIzol® (Invitrogen, USA) as per the manufacturer’s guidelines. cDNA synthesis was performed with the ProtoScript® II First Strand cDNA Synthesis Kit (New England Biolabs) following the provided protocols. RT-qPCR was carried out using the Luna® Universal qPCR Master Mix (New England Biolabs) on a Bio-Rad C1000 Touch™ Thermal Cycler. Bmrp49 was used as the reference gene for normalization. Gene-specific primers for BmToll2, BmToll9−1, and BmToll9−2 were designed with Primer3 based on published sequences, and sequences of all the primers, including the ones for the antimicrobial peptide (AMP) genes, are listed in S3 Table. Gene expression was quantified using the 2-ΔΔCT method.
Results
Identification of virulence genes in the genome of a clinical isolate of S. pneumoniae strain
The genome of S. pneumoniae strain Spn1, a clinical isolate received as a kind gift from Dr. Samir K. Saha (Dhaka Shishu Hospital, Dhaka, Bangladesh), was sequenced in this research. The serotype of Spn1 is 19A. The analysis identified the isolate as S. pneumoniae with the allelic profile aroE_15, ddl_14, gdh_11, gki_9, recP_10, spi_6, and a potential new xpt allele showing 99.8% identity to xpt_1. This allelic combination did not correspond to any known sequence type (ST) in the PubMLST database and was therefore designated as an unassigned (unknown) ST, most closely related to ST9953, ST8789, and ST12899. The genome sequence length of the Spn1 strain is 2097441 bp (2.09 Mb). The number of total genes is 2071, and the GC content is 39.52%. The map of the genome, phylogenetic tree, and cluster of orthologous groups are shown in Fig 1, Fig 2, and S1 Fig, respectively. The genome of Spn1 is comprised of 56 contigs with 2071 genes, and it is closely related to S. pneumoniae strain, CCUG 28588 (Fig 2). The Spn1 strain belongs to a novel sequence type because only 4 out of 7 loci match with ST-0267. Spn1 does not belong to the clonal complex of ST-0267, and it is a triple locus variant, indicating distant relatedness but not direct lineage. Two incomplete prophage regions (S2 Fig) and a plasmid (S3 Fig), harboring a hypothetical protein of 1206 bp length with 99.83% identity with the repUS423 plasmid (NCBI accession number CP003594), (S4 Table) was identified. Certain antimicrobial genes are identified in the Spn1 genome, including genes conferring resistance to macrolides (ermB, mefA, msrD_2), tetracyclines (tetM), chloramphenicol (cat-TC), and fluoroquinolones (patA), validated across multiple AMR databases with 100% coverage (S5 Table, S6 Table) and these findings are consistent with the AMR profile of Spn1 (S2 Table). In the Spn1 genome, the percentage of virulence genes is 24% (31/129). Several virulence genes are identified in the genome of the Spn1 strain, including pivotal adherence factors (pspA, pspC, pavA), toxins (ply), immune evasion proteases (iga, cppA), and nutrient uptake systems (piaA, piuA, psaA) (Table 1). Using CRISPRCasFinder [47], we identified a Type I CRISPR-Cas system in Spn1, comprising a single CRISPR array (position 22,880–22,964) with one spacer and a dedicated cas operon (position 15,076–16,419) encoding two Cas3 proteins. The spacer’s repeat consensus (5’-CTTTTTTTGAAACGTTTCATTTTT-3’) aligns with typical Type I repeats, while the Cas3 proteins confirm functional classification. The spatial separation between the CRISPR array and cas genes is consistent with Type I genomic architecture, suggesting a potentially functional system despite low spacer acquisition activity (S7 Table). The genome sequence data revealed that virulence genes are present in the genome of S. pneumoniae strain, Spn1.
The contents of the feature rings (starting with the outermost ring) are as follows: Ring 1: reverse strand of coding sequence (CDS) features; Ring 2: forward strand of coding sequence (CDS) features; Ring 3: reverse strand ORFs from the primary sequence of Spn1; Ring 4: forward strand ORFs from the primary sequence of Spn1; Ring 5: Contigs; Ring 6 (black): the GC content; Ring 7 (green and purple): the GC skew.
The tree is based on the GBDP phylogenetic analyses retrieved and modified based on the Type (Strain) Genome Server (TYGS). Tree inferred with FastME 2.1.6.1 from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of the GBDP distance formula d5. The numbers above the branches are GBDP pseudo-bootstrap support values >60% from 100 replications, with an average branch support of 71.9%. The tree was rooted at the midpoint and visualized with PhyD3.
S. pneumoniae kills Bombyx larvae when injected into the hemolymph
To examine the pathogenicity of S. pneumoniae Spn1 strain, we used the Bombyx larvae infection model. All the larvae were dead within 24 h of injection of 3.0 × 108 CFU of Spn1 into the hemolymph (Fig 3A), with melanization (blackening) of the cuticle of dead larvae (Fig 3B). No larval death was observed upon injection with 3.0 × 108 CFU of E. coli DH5α or PBS (Fig 3A). The lethal dose (LD50) of Spn1 for silkworm larvae was determined to be 7.8 × 107 CFU. Collection of hemolymph 12 h post-infection (p.i.) revealed melanization or blackening due to increased melanin formation (Fig 4A). Among the prophenoloxidase (ppo) genes, ppo1 and ppo2 expression were significantly upregulated after 6 h and 12 h p.i., respectively (Fig 4B). The total phenoloxidase (PO) enzyme activity in the hemolymph demonstrated a significant increase 12 h p.i. (Fig 4C). S. pneumoniae infects mammals through the lungs, and therefore, we examined the effect of topical application of Spn1 onto spiracles that have openings on the cuticle of the larval body and are connected to tracheal bushes inside. Infection of trachea by Spn1 through topical application onto spiracles of larvae resulted in no death (S4 Fig), but we observed increased melanization of trachea with increased bacterial localization and load (S5 Fig) along with increased tracheal expression of both ppo1 and ppo2 genes 6 h and 12 h p.i. (S6 Fig) in comparison to control larvae injected with PBS. To examine whether other S. pneumoniae strains can kill Bombyx larvae or not, we used three more clinical isolates labeled as Spn2, Spn3, and Spn4. All these strains killed Bombyx larvae within 24 h of infection (S7 Fig). The LD50 value of the S. pneumoniae Spn2, Spn3, and Spn4 strains for Bombyx larvae was 1.14 × 107, 3.55 × 107, and 4.22 × 107 CFU, respectively. These results indicate that Bombyx larvae can be a useful infection model for studying S. pneumoniae pathogenicity.
(A) Percentage survival of B. mori larvae (n = 10) 24 h after injection into hemolymph (blood) with 3.0 × 10⁸ CFU of S. pneumoniae Spn1 or E. coli DH5α suspended in PBS or an equal volume of PBS. The error bar represents the SEM. Data shown are averages of three independent biological experiments. (B) Representative images of larvae after 24 h of injection with PBS, E. coli DH5α, or Spn1.
(A) melanization (blackening) of the larval hemolymph 12 h post-infection (p.i.). (B) RT-qPCR analysis of ppo1 and ppo2 gene expression in the larval fat body after 6- and 12 h of infection with 3.0 × 108 CFU of Spn1 through hemolymph. The reference gene used for normalization was Bmrp49. The error bars represent the SEM. Data generated from at least three independent biological experiments, each with two technical replicates, and the statistical significance was determined via two-way ANOVA that showed expression was significantly higher at 12h compared to 6h (p = 0.0005). (C) Increased phenoloxidase (PO) enzyme activity in hemolymph 12 h post-infection (p.i.). The error bars represent the SEM, and the statistical significance was determined via an unpaired t-test, *p < 0.05.
Reduced viability of circulating hemocytes of Bombyx larvae upon S. pneumoniae infection
The injection of 3.0 × 108 CFU Spn1 into the larval hemolymph led to a notable rise in bacterial count within 9 h p. i. (Fig 5). Since bacterial infection can kill immune cells like the circulating hemocytes, we examined their viability by Trypan blue staining. The viability of circulating hemocytes was reduced to 30% in Spn1-infected larvae compared to the control larvae (Fig 6). The results indicate that the circulating hemocytes of Bombyx larvae can be killed by S. pneumoniae.
Larvae (n = 10) were injected with 3.0 × 108 CFU of Spn1 strain, followed by counting of bacterial numbers in the hemolymph by serial dilution and plating. The error bars represent the SEM. Data shown are averages of three independent biological experiments.
The percentage of viable hemocytes in the larval hemolymph after Spn1 infection (3.0 × 108 CFU) was determined by Trypan Blue staining using hemocytometer. The error bars represent the SEM. Data shown are averages of three independent biological experiments, and the statistical significance was determined via two-way ANOVA. The analysis revealed significant difference between control (PBS) and infected (S. pneumoniae) groups (p < 0.001), along significant effects of time (p < 0.001), supporting that Spn1 infection induces a progressive decline in hemocyte viability with time.
Effects of antibiotics on S. pneumoniae-induced death of Bombyx larvae
According to the Clinical and Laboratory Standards Institute (CLSI) standards [48,49], the S. pneumoniae strain, Spn1 used in this study showed resistance to tetracycline, and erythromycin, but sensitivity towards ampicillin, ceftriaxone, and imipenem as determined by both the antimicrobial resistance (AMR) profile (S2 Table) and the minimal inhibitory concentration (MIC) data (Table 2). Next, we examined the antibiotic sensitivity of Spn1 in vivo using Bombyx larvae. Almost 100% survival was observed for Spn1-infected larvae treated with the highest dose (50 μg g-1 body weight) of ampicillin, ceftriaxone, and tetracycline, whereas 80% and 60% survival was observed with imipenem and erythromycin, respectively (Fig 7A), with a significant reduction of melanization (Fig 7B). Spn1 displayed in vitro resistance to tetracycline and erythromycin, but the Bombyx larvae model showed it was sensitive to these antibiotics in vivo. The effective dosage (ED₅₀) values for the five antibiotics tested in the larvae against Spn1 are listed in Table 2. The findings suggest that the sensitivity of S. pneumoniae to antibiotics can be assessed using the Bombyx larvae infection model.
Larvae (n = 10) were injected with 3.0 × 10⁸ CFU of Spn1 through an abdominal leg, followed by injection of indicated doses of antibiotics through another abdominal leg. (A) Percentage survival of larvae after antibiotic injection 48 h post-infection (p.i.).The error bars represent the SEM. Data are representative of three independent biological experiments. Statistical significance was determined via two-way ANOVA, revealing significant effects of both antibiotic type and dose (p < 0.001), with a strong interaction (p < 0.001). Pairwise comparisons were analyzed using Tukey’s post hoc test. Increasing antibiotic doses markedly enhanced larval survival after S. pneumoniae infection. *Significance levels: *p < 0.05, **p < 0.005. (B) A dose of 50 µg g-1 body weight of all antibiotics except erythromycin showed none or significantly reduced death and melanization 48 h post-infection (p.i.).
S. pneumoniae upregulates homologs of mammalian TLR2, TLR4 and AMP genes in Bombyx larvae
The Toll-like receptor (TLR) family members, TLR2 and TLR4, participate in the innate immune response against S. pneumoniae in mammals by playing critical roles in recognizing and responding to infections by this important pathogen [30,32]. The BmToll2 and BmToll9 genes (BmToll9−1 and BmToll9−2 isoforms) are Bombyx homologs of mammalian TLR2 and TLR4 [34,50]. This study demonstrates that injection of the S. pneumoniae strain, Spn1, into the hemolymph of Bombyx larvae resulted in the increased expression of BmToll2 by more than 35- and 50-fold in the fat body and 6 h and 12 h p.i., respectively, in comparison to the uninfected larvae (Fig 8A). For BmToll9−1, no significant upregulation was observed 6 h p.i., and more than 5-fold increased expression was observed after 12 h p.i. (Fig 8A). BmToll9−2 expression was induced in the fat body by ~20-fold after 6 h, and more than 30-fold after 12 h p.i. (Fig 8A). In the trachea, after hemolymph infection by Spn1, BmToll2 exhibited upregulation of approximately 19-fold and 28-fold at 6 h and 12 h, respectively. In contrast, BmToll9−1 expression increased by approximately 3-fold and 5-fold at 6 h and 12 h of infection, respectively (Fig 8B). No upregulation of BmToll9−2 was observed in the trachea after 6 h of infection with Spn1, but the expression was increased by ~10-fold after 12 h (Fig 8B). Topical application of Spn1 onto the spiracles significantly induced the expression of BmToll2, BmToll9−1, and BmToll9−2 after 12 h of infection in both the fat body and trachea (S8 Fig).
RT-qPCR analysis of BmToll2, BmToll9−1, and BmToll9−2 in fat body (A) and trachea (B) isolated from larvae 6 h and 12 h after injection of 3.0x108 CFU of Spn1 through the hemolymph. The reference gene used for normalization was Bmrp49. The error bars represent the SEM. Data are generated from three independent biological experiments, each with two technical replicates, and the statistical significance was determined via two-way ANOVA. The dashed line represents an expression level of 1.00. (A) For fat body, the overall expression was significantly higher at 12 h compared to 6 h (p = 0.004). (B) For trachea, the overall expression was significantly higher at 12 h compared to 6 h (p = 0.0005).
Next, we examined whether the activation of BmToll2, BmToll9−1, and BmToll9−2 in Bombyx larval fat body and trachea upon infection by S. pneumoniae can upregulate expression of the antimicrobial peptide (AMP) genes, including Bombyx defensin genes whose homologs in mammals are known to be upregulated after S. pneumoniae infection [51,52]. In the larval fat body after Spn1 infection through hemolymph, the expressions of BmdefensinA and BmdefensinB were induced by ~10- and ~100-fold after 6 h and 12 h, respectively, whereas the expressions of Bmcecropin-D1, Bmgloverin-2, and Bmgloverin-3 were induced by ~10-, ~ 2000-, and ~5000-fold after 12 h of infection (Fig 9A). A similar expression pattern of the AMP genes was observed in the trachea after Spn1 infection through hemolymph (Fig 9B). These results indicate that the S. pneumoniae-induced upregulation of the homologs of mammalian TLR2 and TLR4 is evolutionarily conserved in insects like B. mori.
RT-qPCR analysis of different AMP genes in the fat body (A) and trachea (B) isolated from larvae 6 h and 12 h after injection with 3.0 × 10⁸ CFU of Spn1. The reference gene used for normalization was Bmrp49. The error bars represent the SEM. Data are generated from at least three independent biological experiments, each with two technical replicates, and the statistical significance was determined via two-way ANOVA. The dashed line represents an expression level of 1.00. (A) For fat body, two-way ANOVA revealed significant effects of time (p < 0.0001), indicating that with time, expression of genes significantly increased. (B) For trachea, two-way ANOVA revealed significant effects of time (p = 0.0014), indicating with time, expression of genes significantly increased.
Discussion
Among the streptococci, S. pyogenes, a Group A streptococci, is a serious pathogen of humans, and the silkworm infection model has been used to study its pathogenicity [53], but no such studies have been carried out for S. pneumoniae, a human respiratory pathogen. This study applies the Bombyx larvae infection model to investigate the pathogenicity of S. pneumoniae,and we have shown that, similar to mammals, the Toll signaling pathway components, BmToll2 and BmToll9 isoforms, homologs of mammalian TLR2 and TLR4, respectively, are upregulated in the Bombyx larvae upon S. pneumoniae infection.
In this study, the clinical isolate of S. pneumoniae, Spn1, that we used belongs to serotype 19A, that has been reported to cause invasive pneumococcal disease, pneumonia, otitis media, and hemolytic uremic syndrome in various countries [54]. For a successful infection of hosts, S. pneumoniae expresses a battery of virulence genes to compromise the host immunity, and the analysis of the whole genome of the Spn1 strain used in this study revealed several such genes (Table 1). The ply gene that encodes a pore-forming toxin of S. pneumoniae, pneumolysin, is detected in the Spn1 genome. Multiple genes encoding for choline-binding proteins, including cbpG and cbpD are also present that are important for colonization and invasion. The presence of genes encoding enzymes like hyaluronidase and enolase facilitates the spread of Spn1 inside the host. The genes, piaA and piuA, responsible for the sequestering of iron from the host and the manganese-uptake gene, psaA, needed for antioxidant defense are also detected (Table 1). The genes encoding for proteases, including htrA, cppA, and zmpB, that are crucial for host protein degradation, evasion of the host immune system, and essential for virulence, respectively, are also present in the Spn1 genome. The presence of all these virulence genes in the genome of Spn1 confirmed that it is a pathogenic strain of S. pneumoniae.
In this study, we showed that four different clinical isolates of S. pneumoniae can kill silkworm larvae within 24 h (Fig 3, S7 Fig). Kaito et al. reported that a clinical isolate of S. pyogenes required more than 96 h to kill larvae with a dose similar to those used in this study for S. pneumoniae [53]. Experiments conducted at the same time with the same silkworm strain with different strains of S. pyogenes and S. pneumoniae may reveal which strain is more pathogenic. Insect PO activity is increased upon bacterial infection [55]. We observed blackening (melanization) of both hemolymph and cuticle of larvae upon S. pneumoniae infection with an increase of PO activity (Fig 4). The extent of melanization of the cuticle of the dead silkworm larvae after S. pneumoniae infection is visibly much lower (Fig 3, S7 Fig) compared to other pathogenic bacterial infections that we reported previously [13–15]. Reduced melanization after S. pneumoniae infection was also reported in D. melanogaster [38]. We also observed a drop of bacterial load in the hemolymph 3 h post-infection (p.i.) (Fig 5), which could be due to the attachment of bacteria to internal host tissues at the onset of infection, as has been observed with some other pathogenic bacteria in Bombyx larvae [13,16,56]. In mammals, S. pneumoniae infection can kill host cells like red blood cells by producing hydrogen peroxide and toxins [57], and likewise, we observed killing of almost 70% of hemocytes in Bombyx larvae within 3 h of S. pneumoniae infection in our study (Fig 6). It would be worthwhile to examine the mechanism of the death of the larval hemocytes by S. pneumoniae.
The initial investigation on the therapeutic efficacy of antibiotics against human pathogenic bacteria in silkworm larvae was conducted by Hamamoto et al. [12]. No study has been carried out to study the therapeutic effect of antibiotics against any Streptococcus species or the host immune response against it in the Bombyx larvae infection model. Although the three antibiotics (ampicillin, ceftriaxone, imipenem) against which the Spn1 strain showed sensitivity in vitro also showed therapeutic effect (80–90% survival) against the bacterium in vivo in the silkworm larvae infection model (Table 2, Fig 7), tetracycline and erythromycin were exceptions. The whole genome sequence analysis revealed the presence of both tetracycline- and erythromycin-resistant genes (S5 Table, S6 Table) in the Spn1 genome. Against tetracycline and erythromycin, the Spn1 strain showed resistance in vitro (S2 Table, Table 2), however, we observed ~100% and ~60% therapeutic effect against these two antibiotics in Spn1-infected silkworm larvae (Fig 7A). At therapeutic doses, antibiotics reduced the blackening of larvae (Fig 7B), supporting the notion that the reduction of bacterial load by antibiotics led to a decrease in melanization. Our group recently encountered a similar mismatch between in vitro and in vivo antibiotic sensitivity findings using E. coli O157:H7, K. pneumoniae, N. circulans, and K. aerogenes [13–16]. Such a disparity between in vitro and in vivo antibiotic susceptibility data is widely considered a common occurrence [58]. These results underscore the necessity of employing infection models to precisely gauge the antibiotic susceptibility of bacteria that are pathogenic to humans. In this context, Bombyx larvae may serve as a fitting infection model for the initial screening of known antibiotics or biological extracts that are effective against multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains of S. pneumoniae or other human pathogens before validating the results in a mouse model. We have recently shown, for instance, that aqueous extracts from hog plum or Indian gooseberry are capable of rescuing Bombyx larvae infected with K. aerogenes [16].
Antimicrobial peptides (AMPs) contain evolutionarily conserved components of innate immunity in multicellular organisms [59,60]. In insects, the primary sites for the AMP production in response to infection are the fat body tissues that are equivalent to mammalian liver and adipose tissue [61]. Among the AMPs, defensins and gloverins are generally most effective against Gram-positive and Gram-negative bacteria, respectively [62]. Streptococcus pneumoniae enhances the expression of β defensin-2 and β defensin-3 in human lung epithelial cells, with β defensin-2 levels increasing after TLR2 activation in human tracheobronchial epithelial cells [51,52]. Our observations that S. pneumoniae significantly upregulates BmdefensinA and BmdefensinB genes in Bombyx fat body (Fig 9) are consistent with these reports. Vertebrate β defensins are structurally more similar to insect defensins than α defensins and θ defensins, indicating that defensins are evolutionarily conserved [62]. B. mori has eleven Toll genes, and overexpression of BmToll2 induces expression of defensin genes in Bombyx cells in vitro [50,63]. Our observations that BmToll2 expression is increased upon S. pneumoniae infection in Bombyx fat body (Fig 8) are consistent with these reports, and it is plausible that S. pneumoniae-induced expression of defensin genes is BmToll2 dependent. It would be worthwhile to know whether BmToll2 knockdown or knockout can affect the upregulation of defensin genes after S. pneumoniae infection. The Drosophila Toll9 can activate the promoters of defensin and other AMP genes when overexpressed in cultured cells [64], however, no antimicrobial response defect was observed in Drosophila Toll9 mutant [65]. BmToll9 shares structural features with mammalian TLR4 [34]. Mammalian TLR4 is involved in defense against S. pneumoniae [66]. We found that BmToll9 is significantly upregulated in the Bombyx fat body in response to the injection of S. pneumoniae into the hemolymph (Fig 8). S. aureus, a Gram-positive bacterium, also upregulates BmToll9 in the fat body, whereas no such upregulation was observed with E. coli, a Gram-negative bacterium [67]. The function of BmToll9 in response to S. pneumoniae infection warrants further studies.
The major route of transmission of pneumococcus is through direct respiratory droplets in humans. Silkworms breathe through trachea through spiracle, the openings of the tracheal system on the integument of the insect [68]. We found that the topical application of S. pneumoniae onto spiracles resulted in the infection of both trachea and hemolymph concomitant with an increased darkening (melanization) of trachea (S5 Fig), although none of the larvae died, probably due to the low dose of topical application of bacteria. In mice, intranasal inoculation of S. pneumoniae activates the coagulation pathway in the lungs in a TLR2-dependent manner to prevent the spread of the bacteria [69]. There are significant similarities in the cellular mechanisms between blood coagulation and insect hemolymph coagulation [70]. Interestingly, in this study, we found that topical application of S. pneumoniae onto the spiracles resulted not only in the increased melanization of the trachea, indicating localization of hemocytes onto the trachea (S5 Fig) but also upregulation of BmToll2, and BmToll9−2 isoform (S8 Fig). Establishment of BmToll2 and BmToll9 mutant silkworms would conclusively reveal their importance in the upregulation of Bombyx defensins and gloverins genes during pneumococcal infection.
In conclusion, our study shows that the larvae of Bombyx mori can be used as an infection model to screen for antibiotics or unknown biological extracts with antibacterial compound(s) that are effective against S. pneumoniae clinical isolates. We showed that, similar to vertebrates, S. pneumoniae upregulates BmToll2, BmToll9, and Bmdefensins in Bombyx larvae, further strengthening the notion that the model is suitable for studying the pathogenicity of this important pathogen.
Supporting information
S1 Fig. Cluster of orthologous groups (COG) of Streptococcus pneumoniae strain 1, Spn1.
The horizontal axis displays Clusters of Orthologous Gene function type, and the vertical axis is the number of annotated genes.
https://doi.org/10.1371/journal.pone.0341929.s001
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S2 Fig. Schematic representation of the predicted prophages in Streptococcus pneumoniae, Spn1 genome identified using PHASTER.
2 prophage regions have been identified, of which 0 regions are intact, 2 regions are incomplete, and 0 regions are questionable. (https://phaster.ca/batches/BB_09a87038f7). In the Spn1 strain, 2 incomplete prophage regions have been identified. (A) One prophage with 14 CDS extending from 90174 bp to 106620 bp (16.4Kb). This prophage consisted of hypothetical proteins, transposase, integrase, tail protein, and phage-like proteins. (B) Another prophage with 9 CDS extending from 35978 bp to 45261 bp (9.2Kb). This prophage consisted of hypothetical proteins and phage-like proteins.
https://doi.org/10.1371/journal.pone.0341929.s002
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S3 Fig. Circular Map showing the plasmid (repUS43) of Streptococcus pneumoniae, Spn1.
The contents of the feature rings (starting with the outermost ring) are as follows: Ring 1: Forward strand of coding sequence (CDS) features; Ring 2: Contigs; Ring 3 (red): the GC content; Ring 4 (green and purple): the GC skew; Ring 5: forward strand ORFs from the plasmid (repUS43) sequence.
https://doi.org/10.1371/journal.pone.0341929.s003
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S4 Fig. Topical application of S. pneumoniae, Spn1 suspension onto spiracles does not kill Bombyx larvae.
Percentage survival of Bombyx larvae (n = 10) 24 h after topical application of 3.0 × 10⁸ CFU or 3.0 × 10⁹ CFU of Spn1 suspended in PBS or an equal volume of PBS. The error bars represent the SEM. Data generated from three independent biological experiments.
https://doi.org/10.1371/journal.pone.0341929.s004
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S5 Fig. Topical application of S. pneumoniae, Spn1 onto spiracles causes melanization (increased darkening) of Bombyx tracheal bushes.
(A) Darkening of tracheae in larvae after 12 h in response to infection through spiracles. Proliferation of bacteria in hemolymph (B) and trachea (B’) 6 and 12 h post-infection (p.i.) after topical application of bacterial suspension 3.0 × 10⁸ CFU or 3.0 × 10⁹ CFU onto spiracles. The error bars represent the SEM. Data are generated from at least three independent biological experiments, and the statistical significance was determined via two-way ANOVA. For S. pneumoniae proliferation in hemolymph (B) and trachea (B’), two-way ANOVA revealed significant effects of time (p < 0.0001), indicating with time, CFU has significantly increased. The statistical analysis also revealed significant presence of Spn1 in infected larvae as opposed to control (PBS) group (p < 0.0001).
https://doi.org/10.1371/journal.pone.0341929.s005
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S6 Fig. Upregulation of ppo1 and ppo2 in trachea isolated from larvae 6 h and 12 h after injection through hemolymph with 3.0 × 10⁸ CFU of S. pneumoniae, Spn1.
The reference gene used for normalization was Bmrp49. The error bars represent the SEM. Data generated from at least three independent biological experiments, each with two technical replicates, and the statistical significance was determined via two-way ANOVA that showed expression was significantly higher at 12h compared to 6h (p = 0.0228). The dashed line represents an expression level of 1.00.
https://doi.org/10.1371/journal.pone.0341929.s006
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S7 Fig. Three clinical isolates of S. pneumoniae, Spn2, Spn3, and Spn4 kill silkworm larvae.
(A) Percentage survival of Bombyx larvae (n = 10) 24 h post-infection (p.i.) with Spn2 (5.1 × 10⁸ CFU), Spn3 (6.60 × 10⁸ CFU), Spn4 (4.5 × 10⁸ CFU), E. coli DH5α (6.60 × 10⁸ CFU), and PBS. The error bars represent the SEM. Data generated from three independent biological experiments. (B) Images of fifth instar larvae 24 h after injection with PBS, E. coli DH5α, Spn2, Spn3, or Spn4.
https://doi.org/10.1371/journal.pone.0341929.s007
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S8 Fig. Effect of topical application of S. pneumoniae onto spiracles on the expression of BmToll2, BmToll9−1, and BmToll9−2 in Bombyx larvae.
RT-qPCR analysis of BmToll2, BmToll9−1, and BmToll9−2 in the fat body (A) and trachea (B) isolated from larvae 6 h and 12 h after topical application of 3.0x108 CFU of Spn1. The reference gene used for normalization was Bmrp49. The error bars represent the SEM. Data generated from at least three independent biological experiments, each with two technical replicates, and the statistical significance was determined via two-way ANOVA. The dashed line represents an expression level of 1.00. (A) For fat body, the overall expression was significantly higher at 12 h compared to 6 h (p < 0.0001). (B) For trachea, the overall expression was significantly higher at 12 h compared to 6 h (p < 0.0001).
https://doi.org/10.1371/journal.pone.0341929.s008
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S1 Table. Biochemical tests of the S. pneumoniae, Spn1 strain, used in this study.
https://doi.org/10.1371/journal.pone.0341929.s009
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S2 Table. Antimicrobial resistance (AMR) profile of S. pneumoniae, Spn1 strain used in this study.
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S3 Table. Primers used for RT-qPCR in this study.
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S4 Table. RepUS423 plasmid identified in the S. pneumoniae, Spn1 strain used in this study.
https://doi.org/10.1371/journal.pone.0341929.s012
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S5 Table. Annotation of Spn1 genome data using the Rapid Annotation using Subsystem Technology (RAST) server.
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S6 Table. Summary of the antimicrobial resistance genes present in the genome of Streptococcus pneumoniae, Spn1 strain used in this study identified by four different databases.
https://doi.org/10.1371/journal.pone.0341929.s014
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S7 Table. The CRISPR/Cas system of S. pneumoniae, Spn1 strain used in this study.
https://doi.org/10.1371/journal.pone.0341929.s015
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Acknowledgments
We are grateful to Dr. Samir K. Saha of Dhaka Shishu Hospital and Child Health Research Foundation (CHRF), Bangladesh, for sharing with us the S. pneumoniae strain used in this study. We thank Mr. Md. Shah Fazlul Haque of North South University for assistance during research work.
Generative AI statement: The authors declare that no Generative AI was used in the preparation of this manuscript.
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